For time points 2, 6, and 9 months, the number of measurements was n=8, and included measurements taken from dogs A1, A2, A3, and A4. For time points 12, 15, 18, 21, and 28 months, the number of measurements was n=6, and included dogs A2, A3, and A4 (A1 died at the age 11 months).

Bilateral full-field electroretinographic recordings of RPGRIP1-deficient dogs. A: Heterozygous carrier NA1 at the age of 6 months. B: Affected dog A1 at the age of 2, 4, and 9 months. C: Affected dog A2 at the age of 2, 4, 9, and 12 months. D: Affected dog A3 at the age of 2, 4, 9, and 12 months. E: Affected dog A4 at the age of 2, 4, 9, and 12 months. The top two recordings are low and high intensity scotopic responses. The bottom two recordings show photopic responses to light-adapted single flash and 30 Hz flicker stimuli.

Thinning of retinal blood vessel and decrease of retinal thickness

Retinal morphology was monitored by color fundus photographs and by OCT in A2 to A4 for up to 2 years and up to 10 months for A1. For each affected MLHD, color fundus photographs displayed a progressive and dramatic thinning of the retinal vascularization, accompanied by a hyperreflectivity of the tapetal area of the fundus, over a 12 month period (Figure 2).

Figure 2

Fundus photographs in RPGRIP1-deficient dogs. A, B: A1 at the age of 2 and 9 months. C, D: A2 at the age of 2 and 12 months. E, F: A3 at the age of 2 and 12 months. G, H: A4 at the age of 2 and 12 months. There is progressive thinning of the retinal vasculature and an increase in hyperreflectivity of the tapetal area of the fundus.

Images of OCT recordings of RPGRIP1-deficient dogs. The insets show the localization of the 3mm horizontal line scan above the optical nerve head. Notable decrease of the retinal thickness is observed in the affected dog A2 between 5 (A) and 12 months (B). Surprisingly, extreme thinning is seen in the 10-year-old affected dog A5 (C).

Figure 4

Decrease of full retinal thickness over time as observed on OCT scans. Bars (+1 standard deviation) represent retinal thickness measured on retinal sections from dogs of different ages. The Student t-test was performed to compare the mean retinal thickness at different stages of the disease. A p<0.05 was considered significant. The retinal thickness decrease was significant between 2 months and 28 months (p=8.7E07).

Histopathology and apoptosis of photoreceptors

To better evaluate the retinal alterations observed on the OCT images, we sacrificed A6 at the age of 2 months, A1 at the age of 11 months, A2 at the age of 28 months, and A5 at the age of 10 years. The eyes were used for histopathological examination (Figure 5). Examination of hematoxylin- and eosin-stained sections revealed thinning of the outer nuclear layer between the age of 2 and 28 months, reflecting the progressive loss of photoreceptor cells. As seen in dog A5, by the age of 10 years, and as observed by OCT, a thin gliotic retinaremainedvisible on histologic section with a complete lack of the photoreceptor layer.

Immunofluorescence staining of the retina of A6 was performed, using the PNA-FITC lectin and the Rho4D2-FITC antibody. Distinct cone and rod labeling (Figure 6F,H) were observed, suggesting that rods and cones were still present at 2 months of age.

To test whether apoptotic cell death was the cause of retinal thinning of over time, we employed the TUNEL technique to stain sections from A6, A1, and A2 for apoptotic cells. Retina sections from A6, A1, and A2 were positive for TUNEL compared to normal retina. Most of the apoptotic nuclei were detected in the outer nuclear layer, with some nuclei detected in the inner nuclear layer and no signal in the ganglion cell layer (Figure 7).

Discussion

In this study, RPGRIP1-deficient MLHD dogs were monitored clinically to define the optimal therapeutic window for retinal gene therapy. Analysis of the ERG findings showed that as early as 2 months of age, cone function was lost, while rod function was preserved. While cone function was undetectable at this time point, the cone photoreceptors themselves were still present in the retina at this early age. At 9 months of age, both cone and rod functions were undetectable. Interestingly, at this time point, assessment of each dog’s ability to avoid obstacles showed that functional vision is retained up to the age of 11 months. Both OCT and histopathology studies revealed a progressive thinning of the neuroretina over the first 2 years of age. TUNEL assayindicated that the apoptotic photoreceptor cell death was the mechanism of this thinning of the neuroretina.

In contrast to the study by Curtis and Barnett [7], which described what was thought to be a rod-cone dystrophy in the MLHD, and in accordance with the recent findings of Turney et al. [8], our results indicated that the RPGRIP1-deficient MLHD manifested a cone-rod dystrophy. Our ERG findings matched the Turney et al. [8] study. In their study, the ERG of a 6-week-old affected MLHD showed significant reduction of the 30 Hz flicker response.

Although the ERG recordings were undetectable at the age of 9 months in several RPGRIP1-deficient dogs, these same affected dogs still retained some residual functional vision at this same age point. Functional vision persisted in at least one dog up to 14 months of age, at a time when the ERG was undetectable in this animal. This analysis suggests that evaluation both of functional vision using a behavioral test, as well as retinal function, using ERG, will have to be performed to evaluate the efficacy of anyfuture treatment using this model. This is in sharp contrast to what we have observed in gene therapy treatedRPE65−/− dogs, where rescue of retinal function was directly associated to an improvement of functional vision as assessed by behavioral studies [3].

Quantification of TUNEL-positive cells in the outer nuclear layer of affected dogs. Apoptosis analysis was performed with fluorescent microscopy and a 40× objective. Terminal dUTP nick end labeling (TUNEL) positive cells stained with fluorescein were visualized and counted. TUNEL-positive cells in the outer nuclear layer (ONL) were quantified as the percent of the total number of cells in ONL per field. Data are the mean±standard error of the mean from 10 fields of view. The graph represents the average of 3 independent experiments. Months are abbreviated as m.

It was surprising to observe that for the 10-year-old RPGRIP1-deficient dog, not only had the outer retina disappeared but so had the inner retina. An analysis of hematoxylin- and eosin-counterstained sections from dogs between 2 months and 10 years of age did not reveal any inflammatory cells. To our knowledge, this phenomenon of complete neuroretinal degeneration has not been described in any other animal models of LCA or retinitis pigmentosa.

To date, the clinical literature on RPGRIP1-LCA patients clearlydocuments early and severe visual disturbances with nystagmus, abnormal visual acuity, nondetectable ERGs, and fundus features of retinal degeneration [22,23]. The close similarities between the clinical disease characteristics resulting from RPGRIP1 gene mutations in humans and in the dog make this RPGRIP1-deficient MHLD a valuable model for the evaluation of gene therapy.

Recombinant AAV-mediated gene therapy has already been shown successful in the first canine model of LCA, the RPE65−/− dog. This RPGRIP1-deficient model is an attractivealternative LCA canine model in the sense that, in contrast to RPE65, it is not a rod-cone dystrophy but a cone-rod dystrophy and that the cells to target are not the RPE but the photoreceptors. A photoreceptor defect is more difficult to treat than a RPE defect, and a large animal model of such a defect has greatintrinsicvalue.

In conclusion, we have characterized the kinetics of functional and structural changes that occur in RPGRIP1-deficient dogs. The results of this present study suggest a therapeutic strategy that consists of initiating gene therapy as early as possible after birth. Considering the smallsize of the MLHD dogs (120–200 g at birth) and our preliminary surgical data (unpublished results), accurate transvitreal subretinal injection is not possible in dogs younger than 2 months. Successful subretinal delivery of a therapeutic vector will be feasible by 2 months of age and may prevent or delay the loss of rod function. This is a valuable spontaneous animal model that represents a unique and important tool to assess the in vivo efficacy of photoreceptor targeted gene based therapeutic strategies for cone-rod dystrophies.

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